Changes in Acupuncture-Induced Specific Acupoint Neurotransmitters are Possibly Related to Their Physiological Functions in Rats

This study investigated changes in neurotransmitters induced by the application of electroacupuncture (EA) at Zusanli (ST36) and Neiguan (PC6). A total of 30 rats were divided into five groups: sham, ST (EA at bilateral ST36 and ST37), ScT (ST plus previous neurectomy of the bilateral sciatic nerves), ScS (sham plus previous neurectomy of the bilateral sciatic nerve), and PC (EA at bilateral PC6 and PC7). The P2X2 receptor expression was stronger in the sham group than in the ST and PC groups (both p < 0.05) but similar between the sham and ScT groups (p > 0.05). Dopamine levels in the extracellular fluid surrounding the acupoints were higher in the PC group than in the sham and ST groups during the postacupuncture period (both p < 0.05). Glutamate levels in the extracellular fluid surrounding the acupoints were higher in the ST group than in the sham group during the acupuncture period (p < 0.05) and higher in the ST group than in the sham and PC groups during the postacupuncture period (both p < 0.05). Serum adrenaline and noradrenaline levels were higher in the PC group than in the sham, ST, and ScT groups (all p < 0.05). Glutamate levels in the CSF were higher in the ST group than in the sham, ScS, and PC groups (all p < 0.05). GABA levels in the CSF were higher in the ST group than in the sham, ScT, and PC groups (all p < 0.05). EA at ST36 and ST37 and PC6 and PC7 exerted an analgesic effect, EA at PC6 and PC7 can enhance heart function, and EA at ST36 and ST37 modulates the cerebral cortex. However, the study needs an evaluation of direct pain behavior, heart function, and brain function in the future.


Introduction
Acupoints and meridians are the main components of acupuncture. Our previous study reported the specifcity of known acupoints; for example, electroacupuncture (EA) at PC6 can downregulate the production of the adrenaline hormone in the hippocampus and cerebral cortex, whereas EA at ST6 can upregulate its production [1]. Studies have performed the partial least square discriminant analysis to determine the distribution of metabolite profles in the cerebral cortex, hippocampus, hypothalamus, heart, and stomach tissues of rats in sham, ST36, and PC6 groups [1,2]. Te fndings of these studies indicated that acupuncture or EA at acupoints can exhibit the specifcity of those acupoints; however, whether acupoint specifcity can be observed in the environment surrounding those acupoints and drive diferent acupuncture signal transmission remains unclear.
Application of manual acupuncture to the tibialis anterior muscle increased the local extracellular concentrations of adenosine triphosphate, adenosine diphosphate, and adenosine, as determined through high performance liquid chromatography with the microdialysis technique [3]. A study examining dermal microdialysis data reported that the application of EA to Quze (PC3) at 10 Hz for 15 min increased nitric oxide and 3′, 5′ cyclic guanosine monophosphate levels in the Ximen (PC4), thus improving local microcirculation [4]. Te fndings indicated that the application of acupuncture at diferent acupoints can lead to the production of diferent substances or neurotransmitters in tissues surrounding the acupoints. Tese substances or neurotransmitters may be responsible for the efect of acupuncture. Te application of manual acupuncture or EA at acupoints can activate sensory mechanoreceptors and nociceptive receptors in tissues surrounding the acupoints [5]. Most of the acupoints contain many nerve endings and Meissner's corpuscles, Rufni endings, Pacinian corpuscles, and Krause end bulbs that are all encapsulated. Te insertion of an acupuncture needle into an acupoint with Der qi can activate neural and neuroactive components; the collection of activated neural and neuroactive components in skin, muscle, and connective tissues surrounding the inserted needle is referred to as a neural acupuncture unit [6]. Acupuncture induces the release of ATP from the skin, and the released ATP binds to the purinergic receptor of sensory nerves and subsequently conveys the acupuncture signal from the spinal ganglia to higher centers including the spinal cord, brain stem, hypothalamus, and cerebral cortex [7,8]. Taken together, purinergic receptors, sensory nerves, and mechanoreceptors play a crucial role in acupuncture signal transmission. In addition, acupuncture signal transmission includes circular and neural transmission [9]. To determine the specifc physiological functions of acupoints for clinical application, the present study investigated changes in neurotransmitters released in muscle tissues surrounding the acupoints and in the extracellular fuid, peripheral blood, and cerebrospinal fuid (CSF) after the application of acupuncture at the Zusanli (ST36) and Neiguan (PC6).

Materials and Methods
Te present study was to follow the ARRIVE reporting guidelines for experiment animals including species, sex, body weight, and group.

Animals.
Male Sprague-Dawley (SD) rats, weighing 225-250 g, were purchased from BioLASCO Taiwan Co., Ltd. Te rats were raised in a 12 h light/dark environment in the animal center of China Medical University (CMU). Te room temperature and humidity were controlled using an air conditioner at 20°C-24°C and 50%-70%, respectively. Te rats were provided with adequate drinking water and a fxed formulated diet. Animal use was approved by the Institutional Animal Care and Use Committee of CMU, and all animal experiments were performed in accordance with recommendations in the Guide for the Use of Laboratory Animals (National Academy Press). Protocol No. CMUIACUC 2020 279 1, CMUIACUC 2020 279 2, CMUIACUC 2020 279 3).

Groups.
A total of 30 SD rats were randomly divided into fve groups (n � 6 in each group), and that was according to the GraphPad random assignments. (1) In the sham group, acupuncture needles (No. 32 G, Quian Hui, New Taipei City, Taiwan; 0.27 mm in diameter and 13 mm in length) were applied to the subcutaneous bilateral ST36 and ST37 under 2% isofurane anesthesia that were similar to "tong shen cun" in the human body without electrical stimulation for 30 min. (2) In the ST group, acupuncture needles were inserted into bilateral ST36 (cathode) and ST37 (anode) acupoints under 2% isofurane anesthesia, and the needles were applied to an electrical stimulation machine (Trio 300, Japan); electrical stimulation was applied at 2 Hz until a slight muscle contraction was visible for 30 min. (3) In the ScT group, the same method was applied as that in the ST group; however, the bilateral sciatic nerves were cut proximal to the trifurcation 30 min prior to the insertion of acupuncture needles. (4) In the ScS group, the same method was applied as that in the sham group; however, bilateral sciatic nerves were cut proximal to the trifurcation 30 min prior to the insertion of acupuncture needles. (5) In the PC group, the same method was applied as that in the ST group; however, acupuncture needles were inserted into PC6 and PC7.

Sciatic Neurectomy.
Te rats were placed in the sidelying position under 2% isofurane anesthesia. A longitudinal incision was made using a surgical scalpel from the pelvic cavity to the trifurcation of the tibial, common peroneal, and sural nerves to expose the sciatic nerve. Te sciatic nerve was completely transected from proximal to the trifurcation. A sciatic neurectomy was performed 30 min prior to the insertion of acupuncture needles.
2.4. Acupuncture Acupoints. Zusanli (ST36) is located in the posterolateral aspect of the knee joint, approximately 5 mm below the capitulum fbulae. Shangjuxu (ST37) is located 5 mm down from the ST36 of the hind leg of the rats [10,11]. Daling (PC7) is located in the middle of the wrist of the rat's paw. Neiguan (PC6) is located 2 mm above the transverse crease of PC7, between the tendons of the palmaris longus and fexor carpi radialis muscles, and penetrates the fexor digitorum superfcialis [12,13].

Collection of Extracellular
Fluid, Blood, CSF, Muscle, Spinal Cord, and Brain Tissue Samples. First, the rats were anesthetized with 2% isofurane. Subsequently, a needle was inserted into ST36 or PC6, and a microdialysis probe (Eicom, Japan) was implanted between ST36 and ST37 or between PC6 and PC7. Te samples were collected 30 min after the implantation of the probe (baseline, preacupuncture period). Ten, acupuncture needles were inserted into bilateral ST36 and ST37 or PC6 and PC7 (ipsilateral as a pair), and 2 Hz EA was applied for 30 min. Te samples were collected simultaneously during the EA period (acupuncture period). EA stimulation was stopped, and the needles were removed. Subsequently, the samples were collected for 30 min (postacupuncture period). Te perfusion rate was set at 1.5 μL/min. Te samples were placed in a box with ice cubes to prevent protease activation and then stored at −80°C until they were used for the metabolomic analysis of neurotransmitters (Figures 1(a) and 1(b)).
Finally, the rats were deeply anesthetized with 5% isofurane, and the microdialysis probe was removed. A blood sample was collected from the heart, and a CSF sample was collected from the cisterna magna for the metabolomic analysis of neurotransmitters. Furthermore, samples of muscles surrounding the acupoints were collected for Western blot analysis ( Figure 2).

Liquid Chromatography-ElectrosprayIonization-Tandem
Mass Spectrometry Analysis for the Measurement of Neurotransmitters. Before analysis, 30 μL of the extracellular fuid, serum, and CSF samples was completely mixed with 120 μL of methanol through vigorous vertexing. Te sample methanol mixture was centrifuged at 14,000 rpm for 10 min, and 120 μL of the supernatant was collected for drying in a vacuum concentrator. Te dried samples were dissolved in 50 μL of ultrapure water, and 45 μL of the supernatant was collected after centrifugation at 14,000 rpm for 10 min to measure the levels of neurotransmitters through liquid chromatography-electrosprayionizationtandem mass spectrometry (LC/ESI-MS/MS) by using the multiple reaction monitoring/electrospray ionization (ES)+ mode with the Xevo TQ XS system (Waters, Milford, USA, version 4.2).
Neurotransmitters were separated through reverse phase ultra-performance liquid chromatography (UPLC) on the Acquity UPLC BEH C18 1.7 μm column (2.1 mm × 50 mm; Waters, Milford, USA) at 30°C. Te elution was started using 99% mobile phase A (0.1% formic acid in ultrapure water) and 1% mobile phase B (0.1% formic acid in methanol), held at 1% B for 0.5 min, raised to 90% B in 2.5 min, held at 90% B for 0.5 min, and then lowered to 1% B in 0.5 min. Te column was equilibrated by pumping 1% B for 2 min. Te fow rate was set at 0.2 mL/min with an injection volume of 7.5 μL. Mass spectra and chromatograms were acquired in the ES+ mode and processed using Mass Lynx software (Waters, Milford, USA). Neurotransmitters in each sample were determined using liquid chromatography retention plus tandem mass spectrometry and quantifed on the basis of ion mass transitions.

Western Blot Analysis.
Te samples (acupoint muscles including the tibialis anterior muscle in ST36 and ST37 and fexor digitorum profundus muscle in PC6 and PC7 were homogenized at a ratio of 1 : 4 (w/v) in lysis bufer (50 mM Tris, pH 7.4; 250 mM NaCl, 5 mM EDTA; 50 mM NaF; 1 mM Na3VO4; 1% Nonidet P-40, 0.02% NaN 3 ) containing a protease inhibitor cocktail. Te homogenate was centrifuged at 12,000 rpm for 10 min at 4°C. Te supernatant was collected to measure the protein concentration. Subsequently, 30 μg of the sample was separated through 10% polyacrylamide gel electrophoresis and then blotted on a polyvinylidene fuoride membrane. Te membrane was blocked with 5% nonfat milk in TBST bufer for 1 h and then washed with TBST for 5 min three times. Te membrane was then incubated with primary antibodies diluted in phosphate bufered saline under shaking at 4°C overnight. Subsequently, the membrane was incubated with a secondary antibody (1 h at room temperature) under shaking and washed with TBST 5 min three times before and after the addition of the secondary antibody. Proteins were detected using an electrochemiluminescence kit according to the manufacturer's instructions.

Data Preparation and Statistical
Analysis. Data are presented as the mean ± standard error. All statistical analyses were performed using SPSS. Signifcant diferences among the fve groups were analyzed using one-way analysis of variance, followed by the least signifcant diference posthoc test. A p value of <0.05 indicated statistical signifcance.

Efects of EA on Purinergic Receptor, Cytokines, and
Mechanoreceptors: Western Blot Analysis. Te expression of the P2X2 receptor in the acupoint muscle tissues was stronger in the sham group than in the ST and PC groups (both p < 0.05; Figure 3(a)). However, the expression of the P2X2 receptor did not signifcantly difer among the sham, ScT, and ScS groups; between the ST and PC groups; and among the ST, ScT, ScS, and PC groups (all p > 0.05; Figure 3(a)). Te expression of TNF α and IL 10 receptors in the acupoint muscle tissues did not signifcantly difer among the sham, ST, ScT, ScS, and PC groups (all p > 0.05; Figure 3(a)).
Te expression of P2X3, TRPV1, TRPV4, Piezo1, and Piezo2 receptors in the acupoint muscle tissues did not signifcantly difer among the sham, ST, ScT, ScS, and PC groups (all p > 0.05; Figure 3(b)).  Figure 4). Dopamine levels in the extracellular fuid surrounding the acupoints were higher in the PC group than in the ST group during the acupuncture period (p < 0.05; Figure 4). However, dopamine levels were similar among the sham, ScT, ScS, and PC groups and among the sham, ST, ScT, and ScS groups (all p > 0.05; Figure 4). Dopamine levels in the extracellular fuid surrounding the acupoints during the postacupuncture period were higher in the PC group than in the sham, ScT, and ScS groups (all p < 0.05; Figure 4). However, dopamine levels were similar between the ST and PC groups and among the sham, ST, ScT, and ScS groups (all p > 0.05; Figure 4).

Efects of EA on Neurotransmitters in the Extracellular
Noradrenaline levels in the extracellular fuid surrounding the acupoints were similar during the preacupuncture, acupuncture, and postacupuncture periods among the sham, ST, ScT, ScS, and PC groups (all p > 0.05; Figure 4).
c-Aminobutyric acid (GABA) levels in the extracellular fuid surrounding the acupoints were similar during the    Evidence-Based Complementary and Alternative Medicine 5 preacupuncture, acupuncture, and postacupuncture periods among the sham, ST, ScT, ScS, and PC groups (all p > 0.05; Figure 4). Serotonin levels in the extracellular fuid surrounding the acupoints were similar during the preacupuncture, acupuncture, and postacupuncture periods among the sham, ST, ScT, ScS, and PC groups (all p > 0.05; Figure 4).
Glutamate levels in the extracellular fuid surrounding the acupoints were similar among the sham, ST, ScT, ScS, and PC groups during the preacupuncture period (all p > 0.05; Figure 4). Glutamate levels were higher in the ST group than in the sham group during the acupuncture period (p < 0.05; Figure 4) but similar among the sham, ScT, ScS, and PC groups and among the ST, ScT, ScS, and PC groups (all p > 0.05; Figure 4). Furthermore, glutamate levels were higher in the ST group than in the sham and PC groups during the postacupuncture period (both p < 0.05; Figure 4) but similar among the sham, ScT, ScS, and PC groups and among the ST, ScT, and ScS groups (all p > 0.05; Figure 4).
Adrenaline levels in the extracellular fuid surrounding the acupoints were similar during the preacupuncture, acupuncture, and postacupuncture periods among the sham, ST, ScT, ScS, and PC groups (all p > 0.05; Figure 4).

Efects of EA on Neurotransmitters in Peripheral Blood:
Metabolomic Analysis. Serum dopamine levels did not signifcantly difer among the sham, ST, ScT, ScS, and PC groups (all p > 0.05; Figure 5).
Serum adrenaline levels were higher in the PC group than in the sham, ST, and ScT groups (all p < 0.05; Figure 5). Furthermore, serum adrenaline levels did not signifcantly difer among the sham, ST, ScT, and ScS groups and between the ScS and PC groups (all p > 0.05; Figure 5).
Serum noradrenaline levels were higher in the PC group than in the sham, ST, and ScT groups (all p < 0.05; Figure 5). However, serum noradrenaline levels did not signifcantly difer among the sham, ST, ScT, and ScS groups and between the ScS and PC groups (all p > 0.05; Figure 5).

Efects of EA on Neurotransmitters in the CSF: Metabolomic Analysis.
Te dopamine, adrenaline, noradrenaline, and serotonin levels in the CSF did not signifcantly difer among the sham, ST, ScT, ScS, and PC groups (all p > 0.05; Figure 6). Glutamate levels in the CSF were higher in the ST group than in the sham, ScS, and PC groups (all p < 0.05; Figure 6). However, glutamate levels in the CSF did not signifcantly difer among the sham, ScT, ScS, and PC groups and between the ST and ScT groups (all p > 0.05; Figure 6).
GABA levels in the CSF were higher in the ST group than in the sham, ScT, and PC groups (all p < 0.05; Figure 6), whereas GABA levels in the CSF did not signifcantly difer among the sham, ScT, ScS, and PC groups and between the ST and ScS groups (all p > 0.05; Figure 6).
Te aforementioned results are summarized in Tables 1 and 2.

Discussion
Te results of the present study revealed that the expression of P2X2 receptors was stronger in the sham group than in the ST and PC groups and did not signifcantly difer between the sham and ScT groups. Dopamine levels in the extracellular fuid surrounding the acupoints were higher in the PC group than in the sham and ST groups during the postacupuncture period. Glutamate levels in the extracellular fuid surrounding the acupoints were higher in the ST group than in the sham group during the acupuncture period and higher in the ST group than in the sham and PC groups during the postacupuncture period. Furthermore, serum adrenaline and noradrenaline levels were higher in the PC group than in the sham, ST, and ScT groups. Glutamate levels in the CSF were higher in the ST group than in the sham, ScS, and PC groups, and GABA levels in the CSF were higher in the ST group than in the sham, ScT, and PC groups. Te insertion of an acupuncture needle into an acupoint through the skin causes the release of ATP from keratinocytes and fbroblasts; the released ATP then binds to its P2X2 and P2X3 receptors expressed on free nerve endings. Subsequently, the acupuncture signal is transmitted from the peripheral nerve and spinal cord to the brain [7,14]. Te P2X2 and P2X3 receptors are widely expressed on nociceptive sensory neurons and play a vital role in pain transduction. Tese subunits can form homotrimeric P2X2, P2X3, or heterotrimeric P2X2/3 receptors. Each of these receptor subtypes might mediate diferent pathological pain conditions. Te activation of P2X3 leads to rapid desensitization, the stimulation of P2X2 leads to a sustained current, and the activation of P2X2/3 heterotrimers causes intermediate desensitization [15]. A study [16] demonstrated that the intra-arterial infusion of α, β methylene ATP (a selective P2X receptor agonist) caused localized vasoconstriction in the experimental arm. Activation of P2X receptors in the arterial vasculature of the skeletal muscle caused vasoconstriction in both resting and active skeletal muscles [16]. Tese results suggest that EA inhibits the fast synaptic transmission of P2X2 receptors, reducing the vasoconstriction of the skeletal muscle and thus alleviating pain. Te results of the present study indicated that the  bilateral neurectomy of the sciatic nerve reversed the expression of P2X2 receptors reduced by EA at ST36 and ST37. Tis result is consistent with the fnding that acupuncture signals from P2X2 receptors are transmitted from the sensory nerve to the spinal cord and higher centers [7,14]. Taken together, the study initially revoked the capable application of 2 Hz EA at those acupoints for pain relieving treatment.
Our results revealed that the expression of TNF α, IL-10, TRPV1, TRPV4, Piezo1, and Piezo2 did not signifcantly difer among the sham, ST, ScT, ScS, and PC groups. TNF α is a proinfammatory cytokine produced by macrophages in response to infammation, infection, or tissue damage [17]. IL-10 plays an anti-infammatory role and inhibits the production of proinfammatory cytokines, such as IL 1β and TNF α. IL-10 is produced by both immune cells, including T cells, macrophages, and dendritic cells, and nonimmune cells, such as keratinocytes and epithelial cells [18,19]. Te TRPV1 receptor can be activated by capsaicin, nociceptive heat (≥43°C), and camphor and induces pain in the presence of thermal stimuli. Furthermore, the TRPV1 receptor is expressed on the terminals of the small diameter aferent sensory neurons, dorsal root ganglion, spinal cord, and brain [20,21]. TRPV4 plays the role of a transducer in pain caused by osmolarity changes [22]. In addition, TRPV4 can act as a transducer of warm stimuli in the anterior hypothalamus [23]. Te Piezo ion channel is necessary in mechanical transduction processes, such as light touch recognition, proprioception, and vascular blood fow. Piezo1 can regulate red cell volume and shear stress, and Piezo2 is expressed on Merkle cells in response to tough stimulation. Moreover, Piezo2 is required for Merkel cell mechanosensitivity   Evidence-Based Complementary and Alternative Medicine 9 [24,25]. Taken together, the relationship between the application of EA and the expression of TNF α, IL-10, TRPV1, TRPV4, Piezo1, and Piezo2 should be investigated in future studies.
Glutamate is an excitatory neurotransmitter present in the brain and spinal cord and plays a critical role in neuronal cell diferentiation, growth, migration, and survival. Glutamate is involved in many neurological functions, including cognition, memory, neurological network formation, and behavior [26,27]. GABA is a major inhibitory neurotransmitter in the central nervous system, and it regulates the sleep cycle and prevents seizures. GABA is present in diferent brain regions, including the amygdala, hippocampus, hypothalamus, and prefrontal cortex, and modulates excitation and neural synchronization [28]. Te activation of the GABA A receptor reduced neuronal death and provided neuroprotection in focal ischemia mice and rats [29,30]. In Traditional Chinese Medicine (TCM), acupuncture at both ST36 and ST37 belonging to the stomach meridian is applied to treat digestive system disorders, such as abdominal pain and distension [31]. Moreover, acupuncture at ST36 increased motor cortical excitation and reduced motor cortical inhibition [32]. Our previous study reported that the application of EA at bilateral ST36 and ST37 increased the P25 amplitude of somatosensory evoked potentials and inhibited skin sympathetic responses, thus causing cerebral modulation [33]. Acupuncture at ST36 transports substances possibly through a primitive vascular-like system that links between the skin and brain to promote neurogenesis [34]. Taken together, the results suggest that the application of EA at bilateral ST36 and ST37 modulates the excitation of the cerebral cortex, enhances neurological function, and provides neuroprotection. Glutamate from the presynaptic neuron released into the synaptic cleft and combines with N methyl-D-aspartic acid (NMDA) and 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) receptors of postsynaptic neurons can cause the generation of long-term potentiation in the central nervous system. Long-term potentiation plays a critical role in memory and cognitive function, whereas GABA inhibits the propagation of action potential [35]. In addition, metabotropic GABA B receptors can directly afect diferent types of postsynaptic glutamic receptors to modulate the excitability of circuits in brain. In contrast, NMDA receptor activity can also modulate GABAB receptors expression [36], therefore, both glutamate and GABA B play a role of each other's regulation.
Dopamine in the brain mainly originates from dopaminergic neurons in the substantia nigra and ventral tegmental area and regulates cognition, locomotor control, and emotion [37,38]. Peripheral dopamine mainly results from amine precursor uptake and the decarboxylation of cells in the sympathetic nervous system and adrenal glands in response to environmental stress, such as that induced by a high fat diet [37]. Dopamine can reduce renal vascular resistance and increase renal blood and cardiac output in patients with heart failure [39]. Adrenaline can increase left ventricle contraction and cardiac output [40]. Noradrenaline can increase the heart rate and reduce atrioventricular conduction to increase the strength of cardiac contraction, and adrenaline can afect the cardiac muscle and peripheral vessels [41]. In addition, circulating adrenaline causes large increases in blood glucose and energy substrate levels in the skeletal muscles, heart, and brain during a sympathetic response [42]. Acupuncture at both PC6 and PC7 belonging to the pericardium meridian is applied to treat cardiovascular diseases, such as cardiac pain and palpitation, in TCM [31]. Furthermore, the application of EA at PC6 ameliorated myocardial ischemia (MI) in an ischemic heart rat model by mediating the activation of A1 and A2b adenosine receptors [43]. Acupuncture at PC6 increased left ventricular systolic and diastolic function in rats with isoproterenol-induced MI [44]. Taken together, the application of EA at PC6 and PC7 can enhance heart function. Both dopamine and noradrenaline have a vasopressor efect and are used as frst-line agents to correct hypotension in patients with circulatory shock; dopamine is weaker than noradrenaline in vasopressor efect but can increase cardiac output more than noradrenaline. In addition, dopamine can increase global blood fow including renal and hepatosplanchnic blood fow [45].
In TCM, acupuncture is mainly used to treat various diseases in accordance with the principle of meridian theory. For instance, acupuncture at PC6 belonging to the pericardium meridian can treat cardiovascular disease. Similarly, acupuncture at ST36 belonging to the stomach meridian can treat gastric disorders [46]. Te results of the present study demonstrated that application of 2 Hz EA at PC6 and PC7 increased dopamine levels and serum adrenaline and noradrenaline levels in the extracellular fuid surrounding the acupoints. Tese results support meridian theory by indicating that acupuncture at PC6 can treat cardiovascular disease. Moreover, the fndings of the present study indicated that the application of 2 Hz EA at ST36 and ST37 increased glutamate levels in the extracellular fuid surrounding the acupoints and glutamate and GABA levels in the CSF. Acupuncture signal from acupoint and from splanchnic organs is integrated in the central nervous system [46,47]. Moreover, the results suggest that circulating neurotransmitters are involved in transmitting acupuncture signals.
Because neurotransmitter has a clear physiological function of body organs including the heart and nervous system, the present study chooses neurotransmitters, and the present study also defnes acupoint specifcity that acupuncture at the acupoint can produce neurotransmitter changes related to the physiological function of the acupoint. Te present study lacks direct detect painful behaviors as well as heart and brain function, so it can only be said to be an observation of a phenomenon, not direct evidence that EA could modulate pain, heart, and brain function through these receptors and neurotransmitters. Furthermore, study is needed in the future.
Several studies report that isofurane can inhibit mitochondria complex I or presynaptic Ca 2+ infux to inhibit synaptic vesicle exocytosis at nerve terminals. Tese efects of isofurane can afect neurotransmitter release including glutamate and GABA [48,49]. In addition, the rats were operated, the microdialysis probes were implanted, and acupuncture needles were inserted into muscle tissues. Tese procedures were all invasive, all of which may afect possibly the relevant neurotransmitters. Terefore, how to correct for these factors are an important issue. In this study, we deigned a sham group without EA, and acupuncture needles were only inserted into the subcutaneous layer between ST36 and ST37. Te extracellular fuids were collected in the preacupuncture, acupuncture, and postacupuncture periods, and that was similar to the ST and PC groups as possible as to reduce the bias. However, how to seek an anesthetic that does not afect neurotransmitters, use smaller microdialysis probes, and acupuncture that minimizes tissue damage such as laser acupuncture in the future study.

Conclusion
Application of 2 Hz-EA at ST36 and ST37 and PC6 and PC7 reduced the expression of P2X2 receptors in the acupoint muscles, and this efect of EA at ST36 and ST37 could be reversed through the neurectomy of the bilateral sciatic nerves, indicating that the analgesic efect of EA at ST36 and ST37 and PC6 and PC7 is related to sensory aferent nerve transmission. Moreover, the application of 2 Hz-EA at PC6 and PC7 increased dopamine levels in the extracellular fuid surrounding the acupoints and serum adrenalin and noradrenalin levels, indicating that EA at PC6 and PC7 can enhance heart function. In addition, the application of 2 Hz-EA at ST36 and ST37 increased glutamate levels in the extracellular fuid surrounding the acupoints and glutamate and GABA levels in the CSF, indicating that EA at ST36 and ST37 modulates the cerebral cortex to promote and protect neurological function. Tese results are consistent with meridian theory in TCM. However, the present study lacks direct detect painful behaviors as well as heart and brain function, further study is needed in the future.
As this present study is the frst to investigate the efects of EA on alterations of neurotransmitters that might be associated with its physiological functions, extensive research is needed to clarify whether EA can indeed result in analgesic efects and efects on other organs, such as heart and kidney functions. In the same way, we are conducting follow-up research with more critical outcomes that will prove the efects of EA.

Data Availability
Te data supporting the fndings of the study are available from the corresponding authors upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.